Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-09-29 to 2020-10-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tribenzylamine
EC Number:
210-638-3
EC Name:
Tribenzylamine
Cas Number:
620-40-6
Molecular formula:
C21H21N
IUPAC Name:
Tribenzylamine
Test material form:
solid: particulate/powder

Method

Target gene:
Salmonella typhimurium: the strains used contain mutations in the histidine operon
Escherichia coli: the strain used carries a defect in one of the genes for tryptophan biosynthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : liver S9 mix from rats pretreated with ß-Naphthoflavone/Phenobarbital
- method of preparation of S9 mix: The complete S9 Mix was prepared with components: sodium phosphate-buffer 0.5 mL + Rat liver homogenate (S9) 0.1 mL (1st series) or 0.2 mL (2nd series) + Magnesium Chloride/ potassium Chloride Solution (0.4 M/1.64 M) 0.02 mL + Glucose-6-phosphate x 1 H2O, disodium salt 1.61 mg + Nicotinamide adenine dinucleotide phosphate, disodium salt 3.15 mg + ultra-pure water 0.38 mL (1st series) or 0.28 mL (2nd series)
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 20 % S9 in the S9 mix were used. 0.5 mL S9 mix were added per plate with metabolic activation.
- quality controls of S9: metabolic capability
Test concentrations with justification for top dose:
1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate
2nd series: 15.8, 50, 158, 500, 889 µg/plate
The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. Therefore, 5000 µg/plate was chosen as the appropriate maximum test material concentration.
Vehicle / solvent:
- Solvent used: DMSO

- Justification for choice of solvent: The selection of the solvent for this assay was based on the available information from a prelimi-nary solubility test. DMSO showed best Performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

- Justification for percentage of solvent in the final culture medium: Analysis of the historical data and independent experiments of the laboratory, and experience of other research groups showed that the chosen amounts of the selected solvents have no influence on the spontaneous mutation rate of the bacteria used. For this reason, no untreated controls were used.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
other: 4-Nitro-o-phenylenediamine (4-NOPD), 2-Aminoanthracene (2-AA)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
The assay is considered valid if the following criteria were met:
• Regular background growth in the negative control
• The mean number of revertants in the negative (solvent) controls are within the normal range for this test system taking published ranges and the current historical data of the laboratory into account, and
• The positive control chemicals should induce an increase the mean number of revertants above a threshold of 2-fold (TA98, TA100, WP2 uvrA) or 3-fold (TA1535, TA1537) as compared to the concurrent negative controls
• A minimum of five analyzable concentrations, at least three of them without showing signs of toxic effects, evident as a reduction in the number of revertants below a factor of 0.5 compared to the concurrent negative control, should be present.

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA98, TA 100, WP2 uvrA) or 3-fold (TA1535, TA 1537) as compared to the concurrent negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met
Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response.
Statistics:
Tables of individual and mean values were generated by use of a validated, automated data processing program ("Ames Study Manager" and modules by Instem, Stone, Staffordshire, UK, TOXID8004).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate.
- Other confounding effects: No other effects observed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Please refer to "Any other information on results".

Ames test:
- Signs of toxicity : No toxicity to the bacteria was observed with the exception of one occasion in TAI 537 at 5000 ug/plate (without S9 mix).
- Please refer to "Any other information on results" for values.


HISTORICAL CONTROL DATA
- Please refer to "Any other information on results" for values.

Any other information on results incl. tables

1st series:

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
TA98 TA100 TA1535 TA1537 WP2 uvrA
Results without S9
DMSO 36 ± 9 125 ± 13 33 ± 1 14 ± 6 31 ± 5
5 39 ± 3 146 ± 20 46 ± 14 8 ± 4 24 ± 6
15.8 36 ± 12 147 ± 4 68 ± 8 13 ± 6 26 ± 4
50 46 ± 8 133 ± 23 75 ± 7 14 ± 1 22 ± 8
158 39 ± 4 108 ± 4 82 ± 22 11 ± 4 25 ± 4
500 35 ± 6 ME 95 ± 9 ME 75 ± 13 ME 8 ± 3 ME 24 ± 2 ME
1580 38 ± 14ME 101 ± 8 ME 77 ± 11 ME 8 ± 3 ME 19 ± 1 ME
5000 30 ± 2ME 98 ± 6 ME 56 ± 4 ME 6 ± 1 ME 22 ± 3 ME
4-NOPD (20) 225 ± 25
4-NOPD (60) 82 ± 4
NaN3 (2) 1105 ± 27 920 ± 11
NQO (2) 1521 ± 119
Results with S9
DMSO 45 ± 4 119 ± 24 12 ± 3 9 ± 4 36 ± 5
5 38 ± 1 111 ± 5 9 ± 3 13 ± 4 30 ± 7
15.8 44 ± 10 115 ± 6 7 ± 3 11 ± 4 23 ± 4
50 41 ± 5 116 ± 8 11 ± 2 11 ± 4 27 ± 2
158 39 ± 8 104 ± 7 9 ± 3 10 ± 3 26 ± 4
500 36 ± 6 ME 94 ± 4 ME 7 ± 2 ME 9 ± 2 ME 20 ± 10 ME
1580 35 ± 5 ME 81 ± 4 ME 8 ± 3 ME 7 ± 4 ME 20 ± 8 ME
5000 29 ± 8 ME 74 ± 9 ME 9 ± 3 ME 6 ± 1 ME 24 ± 7 ME
2-AA (2) 902 ± 83 1531 ± 51
2-AA (5) 256 ± 23 488 ± 105
2-AA (10.0) 320 ± 32

2nd series:

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
TA98 TA100 TA1535 TA1537 WP2 uvrA
Results without S9
DMSO 42 ± 2 108 ± 7 13 ± 3 16 ± 3 34 ± 4
15.8 35 ± 6 114 ± 29 21 ± 0 20 ± 3 21 ± 4
50 44 ± 7 113 ± 13 22 ± 5 13 ± 4 32 ± 5
158 33 ± 3 90 ± 4 23 ± 1 15 ± 2 35 ± 1
500 30 ± 8 ME 84 ± 8 ME 18 ± 3 ME 12 ± 3 ME 24 ± 6 ME
889 33 ± 8 ME 88 ± 2 ME 16 ± 3 ME 12 ± 3 ME 26 ± 7 ME
4-NOPD (20) 505 ± 23
4-NOPD (60) 108 ± 4
NaN3 (2) 1955 ± 92 1193 ± 76
NQO (2) 2007 ± 88
Results with S9
DMSO 45 ± 6 131 ± 15 16 ± 3 16 ± 3 40 ± 3
15.8 54 ± 16 120 ± 5 18 ± 5 25 ± 2 34 ± 6
50 42 ± 1 144 ± 15 13 ± 5 18 ± 4 36 ± 4
158 53 ± 10 122 ± 25 15 ± 7 18 ± 2 32 ± 7
500 36 ± 2 ME 100 ± 13 ME 10 ± 2 ME 11 ± 2 ME 27 ± 6 ME
889 36 ± 6 ME 105 ± 17 ME 12 ± 1 ME 9 ± 1 ME 25 ± 1 ME
2-AA (2) 579 ± 45 1057 ± 38
2-AA (5) 225 ± 16 191 ± 83
2-AA (10.0) 297 ± 8

E= Precipitation until end of experiment

M= Manual count

Historical Data

The historical data have been obtained in experiments between 01/2019 and 12/2019.

Negative Controls

Strain

TA 98

TA 100

S9 Mix

Without

With

Without

With

Compound

Solvent

Solvent

Solvent

Solvent

Total Plates

424

424

420

416

Number of Values

87

87

86

85

Minimum

8

10

85

93

Maximum

49

52

141

172

Mean

33

39

112

128

Standard Deviation

6.7

7.7

12.5

15.3

Strain

TA 1535*

T A 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Compound

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Total Plates

268

268

272

272

416

416

Number of Values

48

48

49

49

85

85

Minimum

22

9

5

6

24

25

Maximum

45

53

19

16

45

55

Mean

31

28

10

11

32

37

Standard Deviation

5.0

7.2

2.7

2.6

4.5

6.5

 

 

Positive Controls

Strain

TA 98

TA 100

 

S9 Mix

Without

With

Without

With

Compound

DAUN

4-NOPD

2-AA

NaN3

2-AA

Total Plates

90

127

217

215

213

Number of Values

35

52

87

86

85

Minimum

86

68

73

871

354

Maximum

1414

726

5113

2224

5269

Mean

524

446

801

1654

1816

Standard Deviation

337.2

115.1

665.7

281.1

1037.5

 

Strain

T A 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Compound

NaN3

2-AA

9-AA

4-NOPD

2-AA

NQO

2-AA

Total Plates

139

139

62

79

141

213

213

Number of Values

48

48

21

28

49

85

85

Minimum

620

79

104

70

69

386

99

Maximum

1427

273

3715

123

799

2661

729

Mean

947

184

897

99

374

1611

298

Standard Deviation

160.4

49.5

930.4

12.6

198.1

415.9

107.4

 

*ln the present study, TA1535 of a different origin was used which has a generally lower spontaneous mutation frequency as com-pared to the TA1535 strain used to generate the historical control data.

Applicant's summary and conclusion

Conclusions:
It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.
Executive summary:

A study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse mutation test in the absence and presence of a rat liver metabolizing system (S9 mix) according to OECD 471. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pre-treated with ß-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10 % S9 in the 1st and 20 % S9 in the 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate. No toxicity to the bacteria was observed with the exception of one occasion in TAI 537 at 5000 µg/plate (without S9 mix). Under the experimental conditions reported, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.